An improved assay method for fibroblast gelatinolytic enzyme.
A useful gelatinolytic enzyme assay for fibroblasts, utilizing a novel sample preparation method for collagenase with dithiothreitol (DTT) treatment to inactivate endogenous collagenase inhibitors, was developed using soluble fluorescein isothiocyanate (FITC)-labeled gelatin. The substrate, gelatin was prepared by heating commercially available FITC-labeled type I collagen. The denatured collagen was cleaved with purified trypsin and partially purified fibroblast gelatinase, and the digested FITC-fragments were measured fluorometrically. The intensity of the fluorescence was in proportion to the reaction time and enzyme concentration. Both enzyme activities were measurable within the nanogram range of enzyme preparations. The enzyme activity was detected after 4-aminophenylmercuric acetate (APMA) treatment which was completely inhibited by metalloproteinase inhibitors, but not by serine- and cysteine-proteinases' inhibitors. Conditioned media of human periodontal ligament fibroblasts (PLF) and gingival fibroblasts (GF) were separately treated with DTT prior to the enzyme assay, and then the assay was performed in the presence of APMA. The enzyme activities of PLF and GF were 106- and 55-fold higher than those of the conventional gelatinase assay which was carried out without DTT treatment. This assay method allowed the measurement of gelatinolytic enzyme activity when tissue inhibitors of metalloproteinases were present in the fibroblast culture medium.
Otsuka
K
K
Department of Biochemistry, Nihon University School of Dentistry, Japan.
Ohshima
M
M
Kaku
M
M
Kajima
T
T
Fukuoka
M
M
Kaiya
Y
Y
Suzuki
K
K
D001681:Biological Assay; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005470:Fluorometry; D005780:Gelatin / Q000378:metabolism; D018093:Gelatinases / Q000032:analysis*; D005881:Gingiva / Q000166:cytology; D006801:Humans; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D010513:Periodontal Ligament / Q000166:cytology; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D061965:Matrix Metalloproteinase Inhibitors; D015842:Serine Proteinase Inhibitors; D013439:Sulfhydryl Reagents; D019714:Tissue Inhibitor of Metalloproteinases; C010024:4-aminophenylmercuriacetate; D005780:Gelatin; D003094:Collagen; D014357:Trypsin; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D016650:Fluorescein-5-isothiocyanate; D010662:Phenylmercuric Acetate; D004229:Dithiothreitol